Our previous studies have suggested that the covalent binding of acetaldehyde to proteins may be responsible for the hepatotoxicity of ethanol. These proposed studies will continue to explore this possibility and, in this regard, the following hypothesis has been formulated: During ethanol oxidation in the liver, acetaldehyde forms stable adducts via binding to """"""""reactive"""""""" lysine residues of preferential target proteins; and such binding results in impaired function of these proteins, leading eventually to liver cell injury. The initial objective is to clarify the chemistry of stable binding of acetaldehyde to proteins. By using a previously developed HPLC technique, we intend to establish the identities of the modified amino acid (lysine) residues responsible for stable binding and to determine their structures. In addition, we intend to establish the identity of the major acetaldehyde-lysine adduct(s) formed in the liver during ethanol oxidation and develop a reliable assay for detecting this adduct(s).
A second aim will utilize a hepatocyte culture model to study adduct formation and degradation over extended time periods in order to obtain information concerning the turnover of adducts in the liver. Using this model, we will also initiate studies, correlating the presence of adducts with cell viability and hepatocyte function. As an initial attempt to establish a role of stable adducts in liver injury, a third aim will investigate whether differences exist in adduct formation and/or turnover in periportal versus perivenous hepatocytes since the latter are more susceptible to ethanol-induced injury. A fourth objective will be to study the effects of stable acetaldehyde binding on the function of actin and microsomal drug metabolism since both of these models contain essential and reactive lysine residues. These two systems could be potential targets of acetaldehyde binding in the liver, and by accomplishing detailed studies with these two specific systems, we hope to provide the rationale to look for other key targets of binding. Finally, we hope to confirm the presence of stable acetaldehyde-protein adducts in the liver (or other organs) of chronically ethanol-fed rats, and to firmly establish the identity of the """"""""in vivo"""""""" lysine-acetaldehyde adduct. Correlations between evidence of liver injury and the presence of adducts will also be attempted in this model. These proposed studies hopefully will give valuable information concerning the basic molecular mechanisms of alcohol-induced hepatotoxicity.
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